Stimulation of the carotid sinus nerve (CSN) can induce a long term facilitation (LTF) of ventilation which may last minutes to hours. LTF is primarily manifest by an increase in phrenic nerve amplitude rather than respiratory frequency, suggesting it is due to an increase in the magnitude of the inspiratory synaptic drive. LTF it is thought to represent a form of "memory" within the respiratory motor circuit and is believed to stabilize ventilation during episodic hypoxia in sleep. Serotonergic (5-HT) bulbospinal neurons from the raphe obscurus are activated by CSN afferents and are thought to be responsible for the induction of LTF. Raphe neurons possess endogenous pacemaker activity and fire at very low rates. In addition to 5-HT, many of these neurons release the co-transmitters thyrotropin-releasing hormone (TRH) and substance P (SP) in a frequency dependent manner. It has been postulated that raphe neurons expressing co-transmitters act as "pure" serotonergic neurons when endogenously active and that co-transmitters are only released during active stimulation (eg. during carotid sinus nerve stimulation). Within the phrenic motor nucleus 5-HT has two antagonistic effects: an increase in postsynaptic excitability associated with activation of a 5-HT-2A or 5- HT-2C receptor subtype and a reduction in neurotransmitter release from descending premotor respiratory inputs linked to the activation of either a 5-HT-1A or 5-HT-1B receptor subtype. As a result of these antagonistic actions, 5-HT has only a minimal effect on the magnitude of the inspiratory synaptic drive, suggesting that LTF cannot be mediated by 5-HT alone. The fundamental premise of this application is that the co-release of TRH and SP is required for the induction of LTF and that these co- transmitters act by shifting the balance between the antagonistic actions of 5-HT-2A/2C and 5-HT-1A/1B receptors in favor of an increased inspiratory drive. Based on preliminary data and actions of TRH and SP in other parts of the nervous system, we propose that the induction of LTF depends upon the antagonism of presynaptic inhibition mediated by 5-HT- 1A/1B receptors by TRH and the modulation of postsynaptic N-methyl-D- aspartate receptors by SP. These actions of TRH and SP would result in an increased inspiratory synaptic drive. Both TRH and SP have been linked to the activation of a protein kinase C phosphorylation pathway and we also propose that the time course of LTF reflects the lifetime of this second messenger cascade. To test these hypotheses we will use both a spinal cord slice preparation from juvenile rats and labeled premotor respiratory neurons in cell culture from neonatal rats. The experiments proposed in this application will provide new insight into the modulation of neurotransmission within the phrenic motor nucleus and establish a cellular basis for "memory" within the respiratory motor circuit.